D1A root-structure-regulator peptide in Medicago truncatula: Circular dichroism structure-activity studies

Symbioses between nitrogen-fixing rhizobial bacteria and legumes are of great agricultural importance. We have recently-discovered a 15-amino acid peptide-hormone that contributes to root-nodule formation, essential for rhizobium-legume symbiosis. Manual application of these peptide-hormones increases nodule-formation, as does elevating activity of the corresponding gene in Medicago truncatula. The structure/bio-activity relationship of these hormones has however, until now, remained enigmatic. We report structural characteristics essential for the activity of our hormone, dubbed “D1A”. D1A variants, with alterations in key amino-acids, were analysed via ultraviolet circular dichroism (CD), which generates structure-specific spectra. Spectra were compared to existing literature. Reconciliation of structural data with previously-obtained hormonal-activity data suggests a ‘hairpin’ shape to be biologically-relevant, potentially affecting receptor-binding. We speculate D1A possesses a flexible central sequence of amino acids, facilitating this hairpin structure. Structural stabilisation requires interactions between the distal, aromatic amino acids, with natural hormone-structure also requiring proline-hydroxylation. Mono-hydroxylation resulted in hairpin destabilisation, whilst non-hydroxylation resulted in elevated hairpin character.